PROJECT SUMMARY/ABSTRACT Defects in iron-sulfur cluster assembly or delivery underlie many human diseases and aging processes, yet the detailed mechanisms for these processes are still unknown. Organisms have evolved machinery consisting of specialized proteins that operate together to assemble Fe-S clusters efficiently in a way that minimizes cellular exposure to their toxic constituents (iron and sulfide ions). Many of these proteins are dynamic and participate in weak complexes that have resisted structural analysis. We are studying these proteins and their interactions in solution by a combination of NMR spectroscopy, small angle X-ray scattering, chemical crosslinking, isothermal titration calorimetry, and functional biochemical assays. We are applying this approach to address important questions regarding the mechanism of assembly of Fe-S clusters in human mitochondria. We are building on the results of recent X-ray structures that provide a framework for our investigations. Last year, two independent groups published X-ray structures of the (NFS1-ISD11-Acp)2 complex that exhibited very different quaternary structures. To determine if these two different structures exist in solution, our approach is to introduce 19F into NFS1 at a site that is different in the two structures. If two structures are present in solution, we expect to see separate 19F NMR signals for each. By using 2D NMR experiments, we can investigate the local environment of the probe and, if separate signals are observed may be able to determine the lifetimes of each state. To date, all structural and functional studies of the mitochondrial cysteine desulfurase complex have utilized complexes produced by overexpressing the human proteins NFS1 and ISD11 in E. coli cells. The resulting (NFS1-ISD11-Acp)2 complex contains the holo-form of E. coli acyl carrier protein (Acp) in place of human mitochondrial acyl carrier protein (ACP). We will determine structural and functional properties of (NFS1-ISD11-Acp)2 and (NFS1-ISD11-ACP)2 without an acyl chain and with acyl chains of different lengths to identify differences between Acp and ACP and to test the published hypothesis that acyl carrier protein in the cysteine desulfurase complex provides a regulatory link coordinating mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis. Another aim is to characterize the conformational changes in the cysteine desulfurase complex that accompany different steps in cluster assembly and the transfer of the assembled cluster on ISCU to the co-chaperone HSC20. Although it is known that ISCU populates two interconverting conformations in solution (one structured and one intrinsically disordered), the functional roles of these two states remain to be elucidated. By separately labeling Trp residues in ISCU and NFS1 with fluorine, we have determined that 19F NMR enables the observation of different conformational states. These preliminary results set the stage for experiments designed to answer questions about the roles of the structured and dynamic states of ISCU and how assembled clusters are transferred from the cysteine desulfurase complex to HSC20.